Table 1. Summary of mutants’ properties.

All gB constructs were expressed in Sf9 cells infected with recombinant baculoviruses. Expression was assessed by Western blots of infected supernatants using an anti-gB pAb R68. Trimerization was assessed by detection of trimeric form of protein by C226 or DL16 antibodies using P1 refeed samples prepared under mildly denaturing conditions (0.1% SDS, no reducing agent, no boiling). Xa - factor Xa protease cleavage site, His₆ - hexahistidine tag, Strep - streptag.

Construct	Expression	Trimer	C226/DL16
Mutants to stabilize the prefusion conformation
gB773-foldon	+	+	+/+
gB773-foldon-Xa-His6	+	+	+/+
gB773-GSGTGS-foldon-Xa-His6	+	+	+/+
gB773-GGSGGTGGSG-foldon	+	+	+/+
gB773-GGSGGTGGSG-foldon-Xa-His6	+	+	+/+
gB773-foldon-strep	+	+	+/+
gB773-GSGTGS-foldon-strep	+	+	+/+
gB773-GGSGGTGGSG-foldon-strep	+	+	+/+
gB773-GCNt	+	+	+/+
gB773-GSGTGS-GCNt-Xa-His6	+	+	+/+
gB773-GGSGGTGGSG-GCNt-Xa-His6	+	+	+/+
gB773-GSGTGS-cyto(796-904)-His6	+	+	+/+
gB773-GGSGGTGGSG-cyto(796-904)-His6	+	+	+/+
gB730-GSGS-foldon	+	+	+/+
gB730-GSGTGS-foldon-Xa-His6	+	+	+/+
gB730-GGSGGTGGSG-foldon-Xa-His6	+	+	+/+
gB730-GSGTGS-GCNt-Xa-His6	+	+	+/+
gB730-GGSGGTGGSG-GCNt-Xa-His6	+	+	+/+
gB730-GCNt-cyto(821-904)	+	+	+/+
gB730-GCNt-cyto(796-904)	+	+	+/+
gB730-GSGTGS-cyto(796-904)	+	+	+/+
gB730-GSGTGS-cyto(796-904)-His6	+	+	+/+
gB730-GGSGGTGGSG-cyto(796-904)-His6	+	+	+/+
Mutants to destabilize the postfusion conformation
E502A	+	+	+/+
R505A	+	+	+/+
Q507A	+	+	+/+
N511A	+	+	+/+
I391P	+	+	+/+
T394P	+	−	+/−
A504P/R505G/Q507G/N511G (Quad)	+	+	+/+
A504P/R505G/L506F/Q507G/T509F/N511G (Quad-FF)	+	+	+/+
A504P/R505G/L506W/Q507G/T509W/N511G (Quad-WW)	+	+	+/−
L506F	+	+	+/+
T509F	+	+	+/+
I513F	+	+	+/+
L506K	+	+	+/+
R515P	+	+	+/+
L700G/L711G	+	+	+/−
gB670	+	−	+/−
gB678	+	+	+/−
gB683	+	+	+/−
gB689	+	+	+/−
gB700	+	+	+/−
gB684-GGSGGTGGSG-GCNt-Xa-His6	−	N/D	N/D
gB690-GGSGGTGGSG-GCNt-Xa-His6	−	N/D	N/D
gB701-GGSGGTGGSG-GCNt-Xa-His6	−	N/D	N/D